Fig. 3.

Effect of substrate concentration on the rate of 2-hydroxymetronidazole formation by human liver microsomes (Eadie-Hofstee plots). Metronidazole (16 μM–30 mM) was incubated with pooled human liver microsomes (0.05 mg microsomal protein) in 100-μl reaction mixtures at 37 ± 0.1°C, and terminated with 100 μl of methanol containing metronidazole-d4 (internal standard) after 60 minutes. Following precipitation of microsomal protein, an aliquot (10 μl) of the supernatant was analyzed by HPLC/MS via direct injection, respectively, as described under Materials and Methods.